GOAL #1 LMB-100 Combinations. LMB-100 (previously RG7787) is a next generation recombinant immunotoxin (RIT) developed by the Pastan Lab (NCI/ LMB) in collaboration with Roche. This RIT binds to the cancer antigen MSLN delivers a potent bacterial toxin to the cell cytosol. The toxin, a molecularly engineered variant of Pseudomonas exotoxin A, kills cells by irreversibly modifying a critical enzyme in the protein synthesis pathway resulting in a halt in the cell's ability to produce new proteins. This insult triggers apoptosis in many cell types. AIM 1 (on-going): Evaluate efficacy of MSLN-targeted RITs in pancreatic cancer patients. At least 70% of pancreatic adenocarcinomas express the surface antigen MSLN, making these tumors good targets for MSLN-targeted therapies. Based on our previous results, we initiated A Phase Ib/II Study of Mesothelin-Targeted Immunotoxin LMB-100 in Combination with Nab-Paclitaxel in Participants with Previously Treated Metastatic and/ or Locally Advanced Pancreatic Ductal Adenocarcinoma (PI Alewine) to determine the safety, tolerability and efficacy of the LMB-100 + NAB-paclitaxel combination. This trial received IRB approval in 6/2016 and formally opened for accrual 8/2016. The trial has completed accrual and is now closed. Manuscripts are currently in preparation describing our results. We found that the development of anti-drug antibodies against LMB-100 limits effective treatment to 2 cycles. We have opened a new clinical trial testing combination of LMB-100 with the JAK inhibitor tofacitinib. Tofacitinib has been shown by our collaborators (Fitzgerald, Pastan, Onda) to delay anti-drug antibody formation and also to change the immune composition of tumors resulting in increased efficacy of immunotoxin drugs. AIM 2 (ongoing): Determine whether RG7787 or other mesothelin-targeted RITs can boost the effect of immune activating drugs like immune checkpoint inhibitors (ICIs). Pancreatic adenocarcinoma produces an immunosuppressive microenvironment. Killing tumor cells with oncolytic viruses or administering anti-tumor vaccines can cause immune activation. Combining these treatments with ICIs has been demonstrated to produce anti-tumor immune responses in pre-clinical models of pancreatic cancer. We hypothesize that since RG7787 uses a bacterial toxin to kill tumor cells, it may also induce immune activation within the pancreatic cancer microenvironment that could be leveraged to induce an anti-tumor immune response in combination with ICIs. Because our immunotoxins bind only to human (hMSLN) and not to native mouse MSLN (mMSLN), completing this project requires development of a syngeneic mouse pancreatic cancer cell line expressing hMSLN into a mouse strain that will not reject these cells. We have developed such a model in collaboration with the CAPR group at Frederick (Serguei Kozlov, Leidos). This model was reported in abstract at the AACR Annual Meeting 2019. A manuscript describing our new mouse models is in preparation. Experiments using combined ICI and immunotoxin treatment have been initiated and results are pending. AIM3 (Completed): Determine the effect of mesothelin-targeted RIT on tumor cell protein synthesis. RIT inhibits new protein synthesis. The effect of this on individual levels of tumor cell secreted proteins within tumor cells and in the tumor microenvironment has not been previously examined. We used existing athymic nude mouse xenograft model of pancreatic cancer, as well as a new syngeneic model using a cell line co-developed with CAPR to examine this effect. We found that RIT decreases levels of short-lived proteins including many cancer-associated proteins, and tumor secreted factors important in tumorigenic signaling. This data was presented at AACR Annual Meeting 2018 and published in October 2018. A GOAL #2 Understanding MSLN Signaling. MSLN is the target of many therapeutics being tested in clinical trials. It has previously been shown to increase the aggressiveness of pancreatic cancer. It is unknown whether current anti-MSLN therapies inhibit the pro-tumorigenic signaling by MSLN or whether they are just addressing toxic payloads to tumor cells. AIM 1 (ongoing): Determine a phenotype of MSLN loss in pancreatic cancer cells. We found that loss of MSLN impairs ability to pancreatic cancer cells to establish peritoneal metastasis deposits. MSLN loss impaired the establishment of blood vessels to the new tumor deposits. This data was presented at AACR Annual Meeting 2019. A manuscript reporting this finding is currently submitted. We are continuing to characterize the signaling pathways responsible. AIM 2 (ongoing): Determine why MSLN can be detected in the blood of some but not all patients with tumors that make MSLN. We have identified a model system in mice in which to investigate this question. We are continuing to identify the mechanism responsible for the differential secretion of MSLN into blood.